Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Shelter-housed cats show no evidence of faecal shedding of canine parvovirus DNA.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are deoxyriboncucleic acid (DNA) viruses in the taxon Carnivore protoparvovirus 1. Exposure of cats to either CPV or FPV results in productive infection and faecal shedding of virus. Asymptomatic shedding of CPVs by one-third of shelter-housed cats in a UK study suggests that cats may be an important reservoir for parvoviral disease in dogs. The aim of this cross-sectional study was to determine the prevalence of faecal shedding of CPVs in asymptomatic shelter-housed cats in Australia. Faecal samples (n=218) were collected from cats housed in three shelters receiving both cats and dogs, in Queensland and NSW. Molecular testing for Carnivore protoparvovirus 1 DNA was performed by polymerase chain reaction (PCR) amplification followed by DNA sequencing of the VP2 region to differentiate CPV from FPV. Carnivore protoparvovirus 1 DNA was detected in only four (1.8%, 95% confidence interval 0.49-4.53%) faecal samples from a single shelter. Sequencing identified all four positive samples as FPV. Faecal shedding of CPV by shelter-cats was not detected in this study. While the potential for cross-species transmission of CPV between cats and dogs is high, this study found no evidence of a role for cats in maintaining CPV in cat and dog populations through faecal shedding in the regions tested.

Evaluation of a user guidance reminder to improve the quality of electronic prescription messages.

BACKGROUND: Prescribers' inappropriate use of the free-text Notes field in new electronic prescriptions can create confusion and workflow disruptions at receiving pharmacies that often necessitates contact with prescribers for clarification. The inclusion of inappropriate patient direction (Sig) information in the Notes field is particularly problematic. OBJECTIVE: We evaluated the effect of a targeted watermark, an embedded overlay, reminder statement in the Notes field of an EHR-based e-prescribing application on the incidence of inappropriate patient directions (Sig) in the Notes field. METHODS: E-prescriptions issued by the same exact cohort of 97 prescribers were collected over three time periods: baseline, three months after implementation of the reminder, and 15 months post implementation. Three certified and experienced pharmacy technicians independently reviewed all e-prescriptions for inappropriate Sig-related information in the Notes field. A physician reviewer served as the final adjudicator for e-prescriptions where the three reviewers could not reach a consensus. ANOVA and post hoc Tukey HSD tests were performed on group comparisons where statistical significance was evaluated at p<0.05. RESULTS: The incidence of inappropriate Sig-related information in the Notes field decreased from a baseline of 2.8% to 1.8% three months post-implementation and remained stable after 15 months. In addition, prescribers' use of the Notes decreased by 22% after 3 months and had stabilized at 18.7% below baseline after 15 months. CONCLUSION: Insertion of a targeted watermark reminder statement in the Notes field of an e-prescribing application significantly reduced the incidence of inappropriate Sig-related information in Notes and decreased prescribers' use of this field.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus microplus associated with resistance to flumethrin but not to cypermethrin.

A mutation in the domain II S4-5 linker region of the para-sodium channel gene has been associated previously with synthetic pyrethroid (SP) resistance in the cattle tick (Rhipicephalus microplus) in Australia. This is a C→A mutation at nucleotide position 190, which results in a leucine to isoleucine amino acid substitution (L64I). In a survey of 15 cattle tick populations with known SP resistance status, sourced from Queensland and New South Wales in Australia, there was a strong relationship (r=0.98) between the proportion of ticks carrying the L64I homozygous resistant genotype and the survival percentage after exposure to a discriminating concentration of cypermethrin in the bioassay, as expected. However, among populations resistant only to flumethrin, the L64I homozygous genotype was not found. The sequence obtained for a 167 bp region including domain II S4-5 linker in flumethrin-resistant ticks identified a G→T non-synonymous mutation at nucleotide position 214 that results in a glycine to valine substitution (G72V). The frequency of the G72V homozygous genotype in each population was found to be moderately related to the survival percentage at the discriminating concentration of flumethrin in the larval packet test (r=0.74). However, a much stronger relationship between genotype and resistance to flumethrin was observed when the heterozygotes of L64I and G72V were added to the G72V homozygotes (r=0.93). These results suggest that there is an interaction between the two mutations in the same gene, such that flumethrin resistance might be conferred by either two copies of the G72V mutation or by being a L64I and G72V heterozygote.

Linkage confirms canine pkd1 orthologue as a candidate for bull terrier polycystic kidney disease.

Bull terrier polycystic kidney disease (BTPKD) is a Mendelian disorder with many features reminiscent of human autosomal dominant polycystic disease, the latter disease being due to mutations at PKD1 and PKD2 loci. We investigated the role of the canine pkd1 orthologue in BTPKD via linkage analysis of a large kindred in which the disorder is segregating. Twelve microsatellite markers around the canine pkd1 locus (CFA6) were amplified from the genomic DNA of 20 affected and 16 unaffected bull terriers. An additional 28 affected dogs were genotyped at five key microsatellites. A highly significant multi-point LOD score that peaked over the canine pkd1 locus was observed (LOD = 6.59, best two-point LOD score LOD = 6.02), implicating this as the BTPKD locus.

An international parentage and identification panel for the domestic cat (Felis catus).

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex 'core' panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.

The relation between extent of dorsal hippocampal kindling and delayed-match-to-place performance in the Morris water maze.

Dorsal hippocampal kindling impairs subsequent performance on spatial tasks. The relation between this effect and the extent of kindling achieved prior to testing has not been clearly established. Thus, the present study investigated the effects of dorsal hippocampal kindling on performance of a delayed-match-to-place (DMTP) task in the Morris water maze by assessing performance after each of series of different points in the kindling process including 1, 6, 11, and 16 afterdischarges, 1 stage 1 seizure, and 1 stage 5 seizure. We found that kindling produced a deficit that was apparent very early into kindling in terms of both direct swim (by 1 AD) and escape distance (by 6 ADs) measures but that did not clearly change in severity with further kindling. These results illustrate that kindling of even a few localized hippocampal seizures can disrupt spatial cognition and suggest that the mechanisms mediating memory disruption either do not change substantially as kindling progresses or that compensatory processes are engaged across training that mitigate any further kindling-related deteriorations in performance.

A comparison of rate control and rhythm control in patients with atrial fibrillation.

BACKGROUND: There are two approaches to the treatment of atrial fibrillation: one is cardioversion and treatment with antiarrhythmic drugs to maintain sinus rhythm, and the other is the use of rate-controlling drugs, allowing atrial fibrillation to persist. In both approaches, the use of anticoagulant drugs is recommended. METHODS: We conducted a randomized, multicenter comparison of these two treatment strategies in patients with atrial fibrillation and a high risk of stroke or death. The primary end point was overall mortality. RESULTS: A total of 4060 patients (mean [+/-SD] age, 69.7+/-9.0 years) were enrolled in the study; 70.8 percent had a history of hypertension, and 38.2 percent had coronary artery disease. Of the 3311 patients with echocardiograms, the left atrium was enlarged in 64.7 percent and left ventricular function was depressed in 26.0 percent. There were 356 deaths among the patients assigned to rhythm-control therapy and 310 deaths among those assigned to rate-control therapy (mortality at five years, 23.8 percent and 21.3 percent, respectively; hazard ratio, 1.15 [95 percent confidence interval, 0.99 to 1.34]; P=0.08). More patients in the rhythm-control group than in the rate-control group were hospitalized, and there were more adverse drug effects in the rhythm-control group as well. In both groups, the majority of strokes occurred after warfarin had been stopped or when the international normalized ratio was subtherapeutic. CONCLUSIONS: Management of atrial fibrillation with the rhythm-control strategy offers no survival advantage over the rate-control strategy, and there are potential advantages, such as a lower risk of adverse drug effects, with the rate-control strategy. Anticoagulation should be continued in this group of high-risk patients.

Astrocytes attenuate oligodendrocyte death in vitro through an alpha(6) integrin-laminin-dependent mechanism.

Oligodendrocyte (OL) death occurs in many disorders of the CNS, including multiple sclerosis and brain trauma. Factors reported to induce OL death include deprivation of growth factors, elevation of cytokines, oxidative stress, and glutamate excitotoxicity. Because astrocytes produce a large amount of growth factors and antioxidants and are a major source of glutamate uptake, we tested the hypothesis that astrocytes may have a protective role for OL survival. We report that when OLs from the adult mouse brain were initiated into tissue culture, DNA fragmentation and chromatin condensation resulted, indicative of apoptosis. OL death was significantly reduced in coculture with astrocytes, but not with fibroblasts, which provided a similar monolayer of cells as astrocytes. The protection of OL demise by astrocytes was not reproduced by its conditioned medium and was not accounted for by several neurotrophic factors. In contrast, interference with the alpha(6) integrin subunit, but not the alpha(1), alpha(2), alpha(3), alpha(4), alpha(5), or alpha(v) integrin chains, negated astrocyte protection of OLs. Furthermore, a function-blocking antibody to alpha(6)beta(1) integrin reduced the ability of astrocytes to promote OL survival. The extracellular matrix ligand for alpha(6)beta(1) is laminin, which is expressed by astrocytes. Significantly, neutralizing antibodies to laminin-2 and laminin-5 inhibited the astrocyte mediation of OL survival. These results implicate astrocytes in promoting OL survival through a mechanism involving the interaction of alpha(6)beta(1) integrin on OLs with laminin on astrocytes. Enhancing this interaction may provide for OL survival in neurological injury.

Kindling of claustrum and insular cortex: comparison to perirhinal cortex in the rat.

The perirhinal cortex has recently been implicated in the kindling of limbic generalized seizures. The following experiments in rats tested the selectivity of the perirhinal cortex's epileptogenic properties by comparing its kindling profile with those of the adjacent insular cortex, posterior (dorsolateral) claustrum and amygdala. The first experiment examined the kindling and EEG profiles, and found that both the claustrum and insular cortex demonstrated rapid epileptogenic properties similar to the perirhinal cortex, including very rapid kindling rates and short latencies to convulsion. Furthermore, electrical stimulation of all three structures led to a two-phase progression through stage-5 seizures which had characteristics of both neocortical and amygdaloid kindling. In a second experiment rats were suspended in a harness to allow for more detailed documentation of both forelimb and hindlimb convulsions. With this procedure we were able to detect subtle yet unique differences in convulsion characteristics from each of the kindled sites and stage-5 seizure phases. Some of these convulsive parameters were correlated with changes in FosB/DeltaFosB protein and BDNF mRNA expression measured two hours after the last convulsion. Overall, it appears that the perirhinal cortex is not unique in its property of rapid epileptogenesis. Moreover, the posterior claustrum exhibited the fastest kindling and most vigorous patterns of clonus, suggesting that it may be even more intimately associated with the motor substrates responsible for limbic seizure generalization than is the perirhinal cortex.

Interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo.

An acute trauma to the CNS rapidly results in the upregulation of inflammatory cytokines that include interleukin-1 (IL-1). We report here that the levels of several matrix metalloproteinases (MMPs) are also elevated following a corticectomy trauma injury to the mouse CNS. The delayed upregulation of MMPs compared to that for IL-1 suggests the possibility that inflammatory cytokines regulate MMP production in CNS trauma. To resolve this, we developed a method to isolate and maintain highly enriched human fetal neurons or astrocytes in culture and examined the regulation by cytokines of the activity of a subgroup of MMPs, the gelatinases (MMP-2 and -9). While both neuronal and astrocytic cultures displayed comparable MMP-2 activity, as evidenced by gelatin zymography, levels of MMP-9 were proportionately higher in neurons compared to astrocytes. Of a variety of cytokines and growth factors tested in vitro, only IL-1beta was effective in increasing the neuronal expression of MMP-9. Finally, an IL-1 receptor antagonist attenuated the increase of neuronal MMP-9 in culture and abolished the injury-induced increase of MMP-9 in the mouse brain. These results implicate IL-1beta as a key regulator of neuronal MMP-9 in culture and of the elevation of MMP-9 that occurs following mouse CNS trauma.

Comparison of surgical and medical group survival in patients with left main equivalent coronary artery disease. Long-term CASS experience.

BACKGROUND: Combined severe proximal left anterior descending and proximal left circumflex coronary artery disease, or left main equivalent (LMEQ) disease, defines a prognostic high-risk angiographic subset of patients with chronic ischemic heart disease. While numerous observational and randomized clinical trials showed prolonged survival in surgically compared with medically treated patients with left main coronary artery disease, relatively few observational studies compared surgical and medical therapies in patients with LMEQ disease. The present report of 912 patients with LMEQ disease in the Coronary Artery Surgery Study (CASS) Registry extends the originally published 5-year surgical and medical group survival analysis to more than 16 years of follow-up and permits analysis of LMEQ patient subgroups. METHODS AND RESULTS: The CASS Registry contains 912 patients with LMEQ disease, defined as combined stenoses of > or = 70% in the proximal left anterior descending coronary artery before the first septal perforator and proximal circumflex coronary artery before the first obtuse marginal branch, initially treated with either surgical or nonsurgical therapy. The 15-year cumulative survival estimates were 44% for the 630 patients in the surgical group and 31% for the 282 patients in the medical group. Median survival in the surgical group was 13.1 years (12.7 to 14.1 years, 95% confidence limits) compared with only 6.2 years (4.8 to 7.9 years) in the medical group (difference, 6.9 years; P < .0001). Median survival was also significantly longer in the surgical group stratified by age, sex, anginal class, left ventricular (LV) function, and coronary anatomy. However, coronary artery bypass graft (CABG) surgery did not significantly prolong median survival in patient subgroups with (1) normal LV systolic function, even if a significant right coronary artery stenosis (> or = 70%) also was present, and (2) mildly abnormal (LV score, 6 to 10) LV systolic function. The 15-year cumulative survival in patients with normal LV systolic function in the surgical and medical groups was 63% and 54%, respectively. Median survival was > 15 years in both the surgical and medical groups (P = NS). In patients with normal LV systolic function and right coronary artery stenosis > or = 70%, the 15-year cumulative survival was also similar in the surgical and medical groups (63% and 53%, respectively). Median survival was > 15 years in both the surgical and medical groups (P = NS). The 15-year cumulative survival estimates in all subgroups were affected by convergence of the surgical and medical group survival curves caused by a disproportionate increase in late surgical group mortality. Overall, 26% of patients in the medical group ultimately underwent CABG surgery. If all medical group patients had survived long enough, about 65% would be estimated to have had surgery by 15 years. When the CASS Registry patients with LMEQ disease who participated in the randomized trial or who were randomizable were analyzed, CABG surgery did not prolong the 15-year cumulative survival estimates compared with nonsurgical therapy for randomized (71% versus 67%, respectively) and for randomizable patients (62% versus 92%, respectively) with an LV ejection fraction > or = 50%. CONCLUSIONS: This report, which extends follow-up of more than 16 years in CASS Registry patients with LMEQ disease, shows that CABG surgery prolongs life in most clinical and angiographic subgroups. However, median survival was not prolonged by CABG surgery in patients with normal LV systolic function, even if a significant right coronary artery stenosis (> or = 70%) also was present or in patients with an LV ejection fraction > or = 50% who participated in the CASS randomized trial or who were randomizable.

Comparison of surgical and medical group survival in patients with left main coronary artery disease. Long-term CASS experience.

BACKGROUND: Observational and randomized studies designed to compare surgical and medical therapies in patients with left main coronary artery disease (LMCD) have shown that coronary artery bypass graft (CABG) surgery prolongs life in most patients with LMCD. The present report of 1484 patients with LMCD in the Coronary Artery Surgery Study (CASS) Registry extends the originally published 5-year surgical and medical group survival analysis to more than 16 years of follow-up and permits analysis of LMCD patient subgroups. METHODS AND RESULTS: The CASS Registry contains 1484 patients with > or = 50% left main coronary artery stenosis initially treated with either surgical or nonsurgical therapy. The 15-year cumulative survival estimates were 37% for the 1153 patients in the surgical group compared with 27% for the 331 patients in the medical group. Median survival in the surgical group was 13.3 years (12.8 to 13.8 years, 95% confidence limits) compared with only 6.6 years (5.4 to 7.9 years) in the medical group (difference, 6.7 years; P < .0001). Median survival was also significantly longer in the surgical group stratified by age, sex, anginal class, left ventricular (LV) function, coronary anatomy, and the extent of LMCD. However, CABG surgery did not significantly prolong median survival in patient subgroups with (1) left main coronary stenosis of 50% to 59%; (2) normal LV systolic function; (3) normal or mildly abnormal LV systolic function and a right coronary artery stenosis > or = 70%; and (4) a nonstenotic (< or = 70%) right coronary artery. The 15-year cumulative survival for patients with normal LV systolic function in the surgical and medical groups was 42% and 51%, respectively. Median survival was 14.7 years in the surgical group and > 15 years in the medical group (P = NS). In patients with normal LV systolic function and a right coronary artery stenosis > or = 70%, the 15-year cumulative survival rates were also similar in the surgical and medical groups (40% and 48%, respectively). Median survival was 14.3 years in the surgical group and 14.2 years in the medical group (P = NS). The 15-year cumulative survival estimates for all subgroups were affected by convergence of the surgical and medical survival group curves owing to a disproportionate increase in the late surgical group mortality. Overall, 25% of patients in the medical group ultimately underwent CABG surgery. If all medical group patients had survived long enough, about 47% would be estimated to have had surgery by 15 years. CONCLUSIONS: This report, which extends follow-up of more than 16 years in CASS Registry patients with LMCD, shows that CABG surgery prolongs life in most clinical and angiographic subgroups. However, median survival was not prolonged by CABG surgery in patients with normal LV systolic function, even if a significant right coronary artery stenosis (> or = 70%) also was present. These results extend our understanding of the natural history of LMCD and permit a more accurate estimate of long-term surgical and medical group survival.

Five-year angiographic follow-up of factors associated with progression of coronary artery disease in the Coronary Artery Surgery Study (CASS). CASS Participating Investigators and Staff.

OBJECTIVES: The Coronary Artery Surgery Study (CASS) required participants to undergo follow-up angiography at 5 years to identify clinical and angiographic features associated with progression of coronary artery disease. BACKGROUND: The CASS randomized 780 patients at 11 participating clinical centers between an initial strategy of medical therapy versus bypass surgery. Five clinical sites accomplished follow-up angiography in > 50% of their randomized subjects within a 42- to 66-month period after the entry arteriogram (n = 314). METHODS: Qualified clinical site angiographers, using side by side film review, evaluated an average of 13 segments/patient on both arteriograms for initial stenosis severity, morphologic features, lesion location and occurrence of disease progression or occlusion. Progression was defined as further definite narrowing by > or = 15% and occlusion as lesion progression to > or = 98%. Lesions were subcategorized as to whether they were univariate and had or had not been treated with bypass surgery. Multivariate logistic regression analyses were performed. RESULTS: For nonbypassed segments, right coronary artery and left anterior descending artery proximal and midlocations were associated with disease progression. For stenosis-containing segments, the initial severity, a non-left anterior descending artery location and increased treadmill duration predicted progression. Segment occlusion was associated with initial lesion severity, right coronary artery location and subsequent interval myocardial infarction. There were few predictors of progression or occlusion in bypassed arteries, other than initial lesion severity. CONCLUSIONS: Univariate and multivariate associations with lesion progression and occlusion included diabetes, lesion location, elevated cholesterol level, interval infarction and lesion morphology. These angiographic results, collected in a prospective trial, are consistent with known risk factors.